Project Summary. Infectious diseases are a major global threat to human life. Certain individuals, however, carry naturally selected genetic factors which provide essential protection against life-threatening infections. Genetic approaches thus are essential to unravel the basis of host resistance to viral infection and resulting disease. We have shown that host resistance to murine cytomegalovirus (MCMV) infection differs greatly in MA/My and C57L mice. Thus we combined classical genetics with immune cell phenotyping to screen large numbers of MA/My x C57L cross offspring for genetic modifiers of viral immunity. Our whole genome approach detected a quantitative trait locus (QTL) on distal chromosome 4 (dc4) which controls MCMV levels in spleen within days after infection. Two additional QTLs that control host body weight, or NK cells frequency in spleen during infection were separately mapped to the same locus (86.5 cM) on dc4, which hints that it harbors a critical gene or genes essential to NK cells and host resistance to viral infection. Intriguingly, dc4 overlaps a nest of Tumor Necrosis Factor Receptor Superfamily (TNFRSF) member genes which are known to regulate immune cell activities. TNF signaling via TNFRSF members is crucial in lymphoid organogenesis, lymphocyte survival and proliferation, autoimmune dis- ease, cancer and viral infection.!Indeed, several dc4 TNFR-family members and their conserved human coun- terparts have been shown to regulate natural killer (NK) cells in different virus infections. We predict variation in dc4 TNFR-family member gene expression or polymorphism might underlie antiviral immunity or host response differences observed in C57L and MA/My mice during MCMV infection. A broad, long-term objective of this project is to discover the genetic basis of dc4 control of MCMV resistance, weight loss and NK cells during MCMV infection. We will thus combine classical and exploratory genetics approaches to identify and characterize high priority dc4 candidates. In Aim 1, precision genetics will be applied to pinpoint a dc4 QTL in congenic and recombinant congenic mice. Assessment of immune cell responsiveness and MCMV control in key recombinant congenic mice will underpin gene expression screening and selection of high priority dc4 candidates based on positional mapping, allelic differences and expression variation in vital immune cells. Aim 2 examines antiviral NK cell responses, including NK activation, proliferation, survival and trafficking during MCMV infection in dc4- disparate mice. This effect will be further investigated by comparing mixed dc4 donor NK cells responding to MCMV in the same adoptive transfer hosts, followed by analysis of dc4-regulated NK cell-specific gene expres- sion. High priority candidates will be vetted in CRISPR-modified primary T cells developed to assess the effect of dc4/Tnfrsf expression variation or polymorphism in lymphocyte activities following antigen-specific stimulation with plate-bound antibodies, in co-culture assays with antigen presenting cells expressing key ligands, or in in vivo experiments that test OT-I T cells responding to Ova+ pathogens.!